Induction and stabilization of enzymatic activity in microorganisms

ABSTRACT

The present invention is directed to methods for inducing desired activity in enzymes or microorganisms capable of producing the enzymes. The invention is further directed to methods of stabilizing activity in microorganisms. In specific embodiments, the invention provides methods for inducing and stabilizing nitrile hydratase activity, amidase activity, and asparaginase I activity. The invention further provides compositions comprising enzymes or microorganisms having induced and/or stabilized activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 60/763,301, filed Jan. 30, 2006, and U.S. ProvisionalPatent Application No. 60/822,570, filed Aug. 16, 2006, both of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention is generally related to methods for growingmicroorganisms for enzyme production and compositions comprising enzymesor microorganisms having induced and/or stabilized activity. Moreparticularly, the invention relates to methods for inducing a desiredenzyme activity in microorganisms through use of specific growth mediaand to methods for stabilizing desired activity in an enzyme or amicroorganism capable of producing the enzyme.

BACKGROUND OF THE INVENTION

Microorganisms, and their enzymes, have long been utilized asbiocatalysts in the preparation of various products. The action of yeastin the fermentation of sugar to ethanol is an immediately recognizableexample. In recent years, there has been a growing interest in the useof microorganisms and their enzymes in commercial activities notnormally recognized as being amenable to enzyme use. One example is theuse of microorganisms in industrial processes, particularly in thetreatment of waste products.

Nitrile-containing compounds are used in a wide variety of commercialapplications. For example, nitriles are used in the synthesis of manycommercially useful compounds including amines, amides, amidines,carboxylic acids, esters, aldehydes, ketones, imines, and heterocyclics.Nitriles also are used as solvents, as herbicides, and in the synthesisof detergents and antiseptics. One of the more commercially importantnitriles is acrylonitrile, which is used in the production ofacrylamide, acrylic acid, acrylic fibers, copolymer resins, and nitrilerubbers.

The waste streams generated in the production of nitriles often containhigh concentrations of hazardous nitrogen-containing compounds. Forexample, the waste streams can contain nitriles, such as acetonitrile,acrylonitrile, succinonitrile, and fumaronitrile. Further, such wastestreams may also contain hazardous compounds, such as cyanides,acrylamides, acrolein, and cyanohydrins. As hazardous wastes generallycannot be released legally into the environment, methods for treatingwaste streams to remove or remediate one or more hazardous componentsare important in commercial production processes.

One method for treating nitrogen waste streams has been through the useof certain microorganisms that convert nitrile compounds into theircorresponding amides or acids. For example, U.S. Pat. No. 3,940,316 andU.S. Pat. No. 4,001,081 disclose the use of nitrile hydratasemicroorganisms to produce acrylamide from acrylonitrile.

Generally, nitrile converting microorganisms degrade aliphatic nitrilesin a two step reaction involving nitrile hydratase and amidase. In afirst step, nitrile hydratase catalyzes the hydrolysis of the nitrile(or cyanohydrin) to the corresponding amide (or hydroxy acid). In asecond step, amidase catalyzes the hydrolysis of the amide to thecorresponding acid or hydroxy acid. Similarly, some microorganisms havebeen shown to degrade aromatic nitriles by directly converting thesenitriles to their respective acid through the action of nitrilase.

Since the initial reports documenting the potential commercial utilityof the biological conversion of acrylonitrile to acrylamide, the enzymesinvolved in the microbial degradation of nitriles have receivedconsiderable interest. The possibility of enzymatic preparation ofchiral acids (such as hydroxy acids from cyanohydrin precursors) hasalso been a focus of much interest in this field. Despite promisingresults, the various potential applications of the nitrilehydratase/amidase conversion discussed above have not yet been fullyexploited.

Another example of the growing use of microorganisms and their enzymesis in the formation of aspartic acid. Asparaginase I is an enzyme thatcatalyzes the hydrolysis of asparagine to aspartic acid, as shown below:HOOCCHNH₂CONH₂ +H₂O→HOOCCHNH₂CH₂COOH+NH₃

Asparaginase I can be found in bacteria, plants, and many animals;however, as human white blood cells do not possess the necessaryasparagine synthase enzyme, the cells cannot make asparagine. It hasthus been found that asparaginase I can be effective in the treatment ofhuman malignant leukemia. Leukemia cells typically have low levels ofasparagine synthase, the enzyme sometimes being completely absent.Leukemia cells, therefore, generally require an external source ofasparagine. Since asparaginase I converts asparagine to aspartic acid,administering asparaginase I to a patient suffering from leukemiafurther limits the available source of asparagine for the cancerouscells and functions to weaken the cell making them more susceptible tochemotherapeutic treatments. Accordingly, asparaginase I is typicallyadministered to a leukemia patient as part of a combination therapy witha chemotherapeutic agent.

Asparaginase I for use in such treatment is presently obtained from E.coli bacteria (in the form of a heterotetramer) and Erwinia bacteria (inthe form of a homotetramer), but these sources each have disadvantages.For example, the asparaginase I obtained from E. coli is less effectivethan the asparaginase I obtained from Erwinia. However, it is much moredifficult to produce asparaginase I using Erwinia than with E. coli.Further, these sources can result in the presence of Gram-negativetoxins in the isolated enzyme, which is undesirable. Thus, there remainsa need to increase asparaginase I production from a variety ofmicroorganisms while avoiding simultaneous production of gram negativetoxins, which can be harmful.

Stability, which is a key element for a practical biological catalyst,has been a significant hurdle to using nitrile hydratase and/or amidasein many commercial applications. While immobilization and chemicalstabilizing agents are recognized approaches for improving enzymestability, the current immobilization and stabilization techniques arestill in need of further development. Accordingly, there remains a needin the art for method of inducing higher levels of enzymatic activity ina variety of microorganisms, particularly microorganisms capable ofproducing enzymes useful in the degradation of nitrile-containingcompounds. Further, there is also a need for a method to improve thestabilization of key enzymes in the degradation of nitrile-containingcompounds.

BRIEF SUMMARY OF THE INVENTION

The present invention is generally directed to methods for inducing andstabilizing enzymatic activity in microorganisms. The inventionparticularly makes use of nitrile hydratase producing microorganisms forinducing the production of a number of useful enzymes. For example, incertain embodiments, the invention provides methods useful for inducingproduction of nitrile hydratase (particularly at higher levels thanpreviously possible), asparaginase I, and amidase from nitrile degradingmicroorganisms. In further embodiments, the invention provides methodsof improving the stabilization of various enzymes, such as nitrilehydratase, asparaginase I, and amidase. The invention also providesbio-detoxifying catalysts (particularly incorporating enzymes, such asnitrile hydratase and amidase) that can maintain a commercially usefullevel of enzymatic activity over time. The biodetoxifying catalysts areparticularly characterized in that the enzymatic activity of thebiocatalysts can be induced and stabilized by their environment, asdescribed herein.

The present invention is particularly characterized in that the methodsdisclosed herein can be used to induce enzymatic activity that is bothof a level and stability that is useful in a practical biodetoxifyingcatalyst. The invention is further characterized by the ability toinduce higher levels of asparaginase I from microorganisms, including(but not limited to) Gram-positive microorganisms, and to improve thestability of such asparaginase I activity.

The invention is particularly advantageous in that induction andstabilization of the microorganism can be accomplished without therequirement of introducing hazardous nitriles, such as acrylonitrile,into the environment. Previously, it was believed that induction ofspecific enzyme activity in certain microorganisms required the additionof chemical inducers. For example, in the induction of nitrile hydrataseactivity in Rhodococcus rhodochrous and Pseudomonas chloroaphis, it wasgenerally necessary to supplement with hazardous chemicals, such asacetonitrile, acrylonitrile, acrylamide, and the like. Only according tothe present invention, however, has it been surprisingly discovered thathigh enzymatic activity in nitrile hydratase producing microorganismscan by induced and stabilized with the use of non-hazardous mediaadditives, such as amide containing amino acids, and derivativesthereof. More particularly, according to the invention, asparagine,glutamine, or combinations thereof, can be used as inducers with thecomplete exclusion of hazardous chemicals, such as acetonitrile,acrylonitrile, acrylamide, and the like. Thus, the present inventionbeneficially provides safer methods for production of commerciallyuseful enzymes and microorganisms and their use in further methods, suchas for detoxifying waste streams.

In preferred embodiments, the present invention provides for significantincreases in the production and stability of a number of enzymes, andthe microorganisms capable of producing the enzymes, using modifiedmedia, immobilization, and stabilization techniques, as describedherein. For example induction and stabilization can be increased throughuse of media comprising amide-containing amino acids, or derivativesthereof.

In one aspect, the invention comprises a method for growing a nitrilehydratase producing microorganism. The method preferentially comprisesculturing the microorganism in a medium comprising one or more amidecontaining amino acids or derivatives thereof. In specific embodiments,the nitrile hydratase producing microorganism comprises bacteria fromthe genus Rhodococcus. In preferred embodiments, the amide containingamino acids are selected from the group consisting of asparagine,glutamine, or combinations thereof.

In other embodiments, the invention provides a method for inducing adesired enzyme activity in a nitrile hydratase producing microorganism.Preferably, the method comprises culturing the nitrile hydrataseproducing microorganism in a medium comprising one or more amidecontaining amino acids or derivatives thereof. In specific embodiments,the enzyme activity induced by the method comprises nitrile hydrataseactivity, amidase activity, or asparaginase I activity. The methods ofthe invention can comprise further process steps, such as recovering thecultured microorganism having the desired enzyme activity, recovering anenzyme having the desired activity, affixing the microorganism, or cellstherefrom, to a substrate, and cross-linking cells from themicroorganism.

In still further embodiments, the invention particularly providesmethods for stabilizing a desired activity in an enzyme or amicroorganism capable of producing the enzyme. In one embodiment, suchmethods comprise contacting the enzyme, or a microorganism capable ofproducing the enzyme, with one or more amide containing amino acids.

In further embodiments of the invention, the enzyme, or themicroorganism capable of producing the enzyme, can be immobilized. Suchimmobilization can function to affix the enzyme, microorganism, or cellsto a substrate to facilitate ease of handling. In other embodiments,such immobilization can actually function to stabilize the inducedactivity, thus extending the time during which the induced activity canbe utilized. The immobilization can comprise surface attachment of theenzyme, microorganism, or cells to a substrate. Alternately, theimmobilization can comprise at least partially entrapping the enzyme,microorganism, or cells within a substrate or through cross-linkingcells, such as with glutaraldehyde. This beneficially allows forpresentation of an immobilized material with induced activity (e.g., acatalyst) in such a manner as to facilitate reaction of the catalystwith an intended material and recovery of a desired product whilesimultaneously retaining the catalyst in the reaction medium and in areactive mode.

In particular embodiments, the substrate comprises a polymeric material,which is preferably selected from the group consisting of alginate andamide-containing polymers. Other substrate materials are alsoencompassed by the invention. Stabilized activity according to theinvention is preferentially characterized by maintenance of a specificactivity percentage in relation to the initial activity. For example, inone embodiment, the desired activity of the enzyme, or the microorganismcapable of producing the enzyme, is stabilized such that the desiredactivity after a time of at least 30 days at a temperature of about 25°C. is maintained at a level of at least about 50% of the initialactivity exhibited by the enzyme or the microorganism capable ofproducing the enzyme.

In still further embodiments, the invention provides methods forpreparing an enzyme or microorganism having a specific enzymaticactivity. For example, in one embodiment, the invention provides methodsfor preparing an enzyme or microorganism having nitrile hydrataseactivity. In particular, the method comprises inducing nitrile hydrataseactivity in a microorganism by culturing the microorganism in a mediumcomprising one or more amide containing amino acids or derivativesthereof, and recovering the enzyme or microorganism having nitrilehydratase activity. In further embodiments, the invention providesmethods for preparing an enzyme or microorganism having asparaginase Iactivity or amidase activity.

The invention is still further characterized by the ability to multiplyinduce enzymatic activity in microorganisms such that themicroorganisms, or the enzymes produced thereby, are capable ofdegrading a plurality of compounds. Thus, in one embodiment, theinventive method comprises multiply inducing nitrile hydratase activitytoward a plurality of nitrile containing compounds in a microorganism byculturing the microorganism in a medium comprising one or more amidecontaining amino acids or derivatives thereof. Optionally, the methodfurther comprises recovering the enzyme or microorganism having nitrilehydratase activity for degrading a plurality of nitrile containingcompounds. Of course, in further embodiments, the invention providesmethods of multiply inducing other types of activity.

In another aspect, the present invention provides a novel compositionthat is particularly useful in the methods of the invention, as well asfor the production of various devices, such as biofilters. In oneembodiment, the composition of the invention comprises: (a) a nutrientmedium comprising one or more amide containing amino acids, orderivatives thereof, (b) one or more enzyme-producing microorganisms;and (c) one or more enzymes. Preferably, the enzymes are selected fromthe group consisting of nitrile hydratase, amidase, asparaginase I, andcombinations thereof. In further embodiments, the one or moremicroorganisms comprise bacteria selected from the group consisting ofgenus Rhodococcus, genus Brevibacterium, and combinations thereof. Inpreferred embodiments, the one or more microorganisms can be at leastpartially immobilized on a substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is particularly described in reference to thefollowing figures; however, such figures are provided to illustrate onlypreferred embodiments of the invention, and the invention is notintended to be limited thereto.

FIG. 1 is a graphical illustration of the stabilizing effect on nitrilehydratase activity provided by immobilization in calcium alginate in oneembodiment according to one method of the invention;

FIG. 2 is a graphical illustration of the stabilizing effect on nitrilehydratase activity provided by immobilization in polyacrylamide in oneembodiment according to one method of the invention;

FIG. 3 is a graphical illustration of the stabilizing effect on nitrilehydratase activity provided by immobilization in hardened,polyethyleneimine cross-linked calcium alginate or polyacrylamide infurther embodiments according to one method of the invention;

FIG. 4 is a graphical illustration of the stabilizing effect on nitrilehydratase activity provided by immobilization through glutaraldehydecross-linking according to one method of the invention; and

FIG. 5 is a graphical illustration of the asparaginase I activity inRhodococcus sp. DAP 96253 cells induced with asparagine according to oneembodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to specific embodiments of the invention and particularly tothe various drawings provided herewith. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. As used in the specification, and in the appended claims,the singular forms “a”, “an”, “the”, include plural referents unless thecontext clearly dictates otherwise.

The present invention arises from the surprising finding that specificenzymatic activity can be induced and stabilized through addition ofamide containing amino acids, or derivatives thereof, to a culture orcomposition comprising specific enzymes or microorganisms capable ofproducing such enzymes. Moreover, further stabilization can be achievedthrough immobilization methods, such as affixation, entrapment, andcross-linking. In specific embodiments, methods are provided forinducing higher levels of nitrile hydratase and amidase from nitriledegrading microorganisms (including nitrile hydratase producingmicroorganisms) and of improving the stability of nitrile hydratase.

The inventive methods described herein also provide for further methodsuseful in the detoxification of compositions containing nitrilecompounds through conversion of the nitrile moieties into amides and/oracids. Such methods may be used to remove unwanted nitrile compoundsfrom various compositions, such as waste streams produced frommanufacturing and production facilities.

The present invention generally provides methods for culturingmicroorganisms, preferentially inducing specific enzymatic activity.Preferably, the methods comprise culturing the microorganisms in amedium comprising one or more amide containing amino acids, orderivatives thereof In preferred embodiments, the amide containing aminoacids are selected from the group consisting of asparagine, glutamine,derivatives thereof, or combinations thereof. For example, theamide-containing amino acids may include natural forms of asparagines,anhydrous asparagine, asparagine monohydrate, natural forms ofglutamine, anhydrous glutamine, and/or glutamine monohydrate, each inthe form of the L-isomer or DL-isomer.

The concentration of the amide containing amino acids, or derivativesthereof, in the medium can vary depending upon the desired end result ofthe culture. For example, a culture may be carried out for the purposeof producing microorganisms having a specific enzymatic activity. Inother embodiments, a culture may be carried out for the purpose offorming and collecting a specific enzyme from the culturedmicroorganisms. In yet further embodiments, a culture may be carried outfor the purpose of forming and collecting a plurality of enzymes havingthe same or different activities and functions.

The amount of the amide containing amino acids, or derivatives thereof,added to the growth medium or mixture can generally be up to about10,000 ppm (i.e., about 1% by weight) based on the overall weight of themedium or mixture. The present invention is particularly beneficial,however, in that enzyme activity can be induced through addition of evenlesser amounts. For example, in certain embodiments, the concentrationof the amide containing amino acids, or derivatives thereof, is in therange of about 50 ppm to about 5,000 ppm, about 100 ppm to about 3,000ppm, about 200 ppm to about 2,000 ppm, about 250 ppm to about 1500 ppm,about 500 ppm to about 1250 ppm, or about 500 ppm to about 1000 ppm.

Preferably, the amide containing amino acids, or derivatives thereof,are added to a nutritionally complete media. A suitable nutritionallycomplete medium generally is a growth medium that can supply amicroorganism with the necessary nutrients required for its growth,which minimally includes a carbon and/or nitrogen source. One specificexample of a useful medium is the commercially available R2A agarmedium, which typically consists of agar, yeast extract, proteosepeptone, casein hydrolysate, glucose, soluble starch, sodium pyruvate,dipotassium hydrogenphosphate, and magnesium sulfate. Another example ofa nutritionally complete liquid medium useful according to the presentinvention is Yeast Extract Malt Extract Agar (YEMEA), which consists ofglucose, malt extract, and yeast extract (but specifically excludesagar). Of course, any nutritionally complete medium useful in the artcould be used according to the present invention, the above media beingdescribed for exemplary purposes only.

In further embodiments, the methods of the invention can comprise theuse of further additives to the nutritionally complete media. Typically,the other supplements or nutrients useful according to the invention arethose useful for assisting in greater cell growth, greater cell mass, oraccelerated growth. For example, in one embodiment, the nutritionallycomplete medium can comprise a carbohydrate source in addition to anycarbohydrate source already present in the nutritionally completemedium.

As described above, most media typically contain some content ofcarbohydrate (e.g., glucose); however, according to the presentinvention, it can be useful to include an additional carbohydratesource. The type of excess carbohydrate provided can depend upon thedesired outcome of the culture. For example, in specific embodiments,the addition of carbohydrates, such as maltose or maltodextrin, has beenfound provide for improved induction of asparaginase I.

In another embodiment, cobalt, or a salt thereof, can be added to themixture or media. For example, the addition of cobalt (e.g., cobaltchloride) to the media can be particularly useful for increasing themass of the enzyme produced by the cultured microorganisms. In certainembodiments, cobalt, or a salt thereof, can be added to the culturemedium such that the cobalt concentration is an amount up to about 100ppm. Preferably, cobalt is present in a concentration of about 5 ppm toabout 100 ppm, about 10 ppm to about 75 ppm, about 10 ppm to about 50ppm, or about 10 ppm to about 25 ppm.

In yet further embodiments, urea, or a salt thereof, can be added to themixture or media. In certain embodiments, urea, or a salt thereof, canbe added to the culture medium such that the urea concentration is in anamount up to about 10 g/L. Preferably, urea is present in aconcentration of about 5 g/L to about 100 g/L, about 10 g/L to about 75g/L, about 10 g/L to about 50 g/L, or about 10 g/L to about 25 g/L. Inspecific embodiments, urea is present in a concentration of about 7.5g/L.

The medium may also include further components without departing fromthe present invention. For example, other suitable medium components mayinclude commercial additives, such as cottonseed protein, maltose,maltodextrin, and other commercial carbohydrates.

The present invention is particularly characterized in that inductionand stabilization of enzymes, and microorganisms capable of producingthe enzymes, can be achieved without the need for hazardous nitriles. Aspreviously pointed out, the induction of many types of enzyme activity,such as nitrile hydratase activity, has traditionally includedsupplementation with nitriles, such as acetonitrile, acrylonitrile,succinonitrile, and the like. Moreover, if multiple induction wasdesired (i.e., induction of activity in a single enzyme to degrade twoor more types of nitriles), it was generally necessary to include two ormore types of hazardous nitriles. The present invention, particularlyarising from the use of amide containing amino acids, and derivativesthereof, as enzymatic inducers eliminates the need for hazardouschemicals to facilitate single or multiple enzymatic induction.Particularly, the present invention is beneficial in that multipleinduction is possible through the use of amide containing amino acids,or derivatives thereof, in the culture medium or mixture. Again, this isespecially surprising as multiple nitrile compounds were previouslyrequired in the culture medium to induce enzyme activity toward two ormore nitrile compounds. However, the present invention achieves thisparticularly useful characteristic through the use of completely safeamide containing amino acids. Thus, the present invention isparticularly useful for preparing an enzyme or microorganism havingactivity for degrading a plurality of nitrile containing compounds.Moreover, the methods of the invention provide the ability to detoxify avariety of nitriles or amides, such as nitriles having a single C≡Nmoiety, dinitriles (compounds having two C≡N moieties), or compoundshaving multiple nitrile moieties (e.g., acrolein cyanohydrin). Suchenzymes, or microorganisms, are herein referred to as being multiplyinduced.

While the present invention eliminates the need for hazardous chemicalsfor enzyme activity induction, the use of such further inducers is notexcluded. For example, in specific embodiments, one or more nitrilescould be used to assist in specific activity development. For example,media supplemented with succinonitrile and cobalt can be useful forinduction of asparaginase I activity. However, the use of nitriles isnot necessary for induction of asparaginase I activity. Rather, whilethe use of nitriles and other hazardous chemicals is certainly notpreferred according to the invention, in specific embodiments, such useis possible.

A variety of microorganisms can be cultivated for use according to thepresent invention. Generally, any microorganisms capable of producingenzymes having useful activity, as described herein, can be used in theinvention. In particular embodiments, the microorganisms usefulaccording to the invention comprise microorganisms capable of producingnitrile hydratase.

As used herein, nitrile hydratase producing microorganisms are intendedto refer to microorganisms that, while generally being recognized asbeing capable of producing nitrile hydratase, are also capable ofproducing one or more further enzymes. As further described herein, mostmicroorganisms are capable of producing a variety of enzymes, suchproduction often being determined by the environment of themicroorganism. Thus, while microorganisms for use according to theinvention may be disclosed as nitrile hydratase producingmicroorganisms, such language only refers to the known ability of suchmicroorganisms to produce nitrile hydratase and does not limit themicroorganisms to only the production of nitrile hydratase. On thecontrary, a nitrile hydratase producing microorganisms useful accordingto the invention is a microorganism capable of producing at leastnitrile hydratase (i.e., is capable of producing nitrile hydratase ornitrile hydratase and one or more further enzymes).

A number of nitrile hydratase producing microorganisms are known in theart. For example, bacteria belonging to the genus Nocardia [see JapanesePatent Application No. 54-129190], Rhodococcus [see Japanese PatentApplication No. 2-470], Rhizobium [see Japanese Patent Application No.5-236977], Klebsiella [Japanese Patent Application No. 5-30982],Aeromonas [Japanese Patent Application No. 5-30983], Agrobacterium[Japanese Patent Application No. 8-154691], Bacillus [Japanese PatentApplication No. 8-187092], Pseudonocardia [Japanese Patent ApplicationNo. 8-56684], and Pseudomonas are non-limiting examples of nitrilehydratase producing microorganisms that can be used according to theinvention.

Further, specific examples of microorganisms useful according to theinvention include, but are not limited to, Nocardia sp., Rhodococcussp., Rhodococcus rhodochrous, Klebsiella sp., Aeromonas sp., Citrobacterfreundii, Agrobacterium rhizogenes, Agrobacterium tumefaciens,Xanthobacter flavas, Erwinia nigrifluens, Enterobacter sp., Streptomycessp., Rhizobium sp., Rhizobium loti, Rhizobium legminosarum, Rhizobiummerioti, Candida guilliermondii, Pantoea agglomerans, Klebsiellapneumoniae subsp. pneumoniae, Agrobacterium radiobacter, Bacillussmithii, Pseudonocardia thermophila, Pseudomonas chloroaphis,Pseudomonas erythropolis, Brevibacterium ketoglutamicum, Rhodococcuserythropolis, and Pseudonocardia thermophila. In particularly preferredembodiments, microorganisms used according to the invention compriseRhodococcus sp. DAP 96253 and DAP 96255 and Rhodococcus rhodochrous DAP96622.

In further embodiments, microorganisms useful according to the inventioncan also include transformants. In particular, the transformants can beany host wherein a nitrile hydratase gene cloned from a microorganismknown to include such a gene, is inserted and expressed. For example,U.S. Pat. No. 5,807,730 describes the use of Escherichia coli as a hostfor the MT-10822 bacteria strain (FERM BP-5785). Of course, other typesof genetically engineered bacteria could be used according to theinvention so long as the bacteria are capable of producing one or moreenzymes useful in the invention, as described herein.

Not all species within a given genus exhibit the same type of enzymeactivity and/or production. Thus, it is possible to have a genusgenerally know to include strains capable of exhibiting a desiredactivity but have one or more species that do not generally exhibit thedesired activity. Thus, in still further embodiments, hostmicroorganisms can include strains of bacteria that are not specificallyknown to have the desired activity but are from a genus known to havespecific strains capable of producing the desired activity. Such strainscan have transferred thereto one or more gene useful to cause thedesired activity. Non-limiting examples of such strains includeRhodococcus equi and Rhododoccus globerulus PWD1.

Enzymatic activity, as used herein, generally refers to the ability ofan enzyme to act as a catalyst in a process, such as the conversion ofone compound to another compound. Likewise, the desired activityreferred to herein can include the activity of one or more enzymes beingactively expressed by one or more microorganisms.

In certain embodiments, activity can be referred to in terms of “units”per mass of enzyme or cells (typically based on the dry weight of thecells, e.g., units/mg cdw). A “unit” generally refers to the ability toconvert a specific content of a compound to a different compound under adefined set of conditions as a function of time. In specificembodiments, 1 “unit” of nitrile hydratase activity can relate to theability to convert 1 μmol of acrylonitrile to its corresponding amideper minute, per milligram of cells (dry weight) at a of pH 7.0 and atemperature of 30° C. Similarly, 1 unit of amidase activity can relateto the ability to convert 1 μmol of acrylamide to its corresponding acidper minute, per milligram of cells (dry weight) at a pH of 7.0 and atemperature of 30° C. Further, 1 unit of asparaginase I activity canrelate to the ability to convert 1 μmol of asparagine to itscorresponding acid per minute, per milligram of cells (dry weight) at apH of 7.0 and a temperature of 30° C.

In preferred embodiments, the present invention is particularlycharacterized by the ability to induce a desired activity that isgreater than possible using previously known methods. For example, inone embodiment, the invention allows for inducing nitrile hydrataseactivity in a nitrile hydratase producing microorganism that is greaterthan or equal to the activity produced in the same microorganism byinduction with a nitrile containing compound. In preferred embodiments,the nitrile hydratase activity produced is greater than the activityproduced in the same microorganism by induction with a nitrilecontaining compound. For example, the nitrile hydratase activityproduced according to the methods of the invention can be at least 5%greater than the activity produced in the same microorganism byinduction with a nitrile containing compound. Preferably, the nitrilehydratase activity produced according to the methods of the invention isat least 10%, at least 12%, or at least 15% greater than the activityproduced in the same microorganism by induction with a nitrilecontaining compound.

The microorganisms useful according to the invention can be selectedfrom known sources (such as those described above) or can comprise newlyisolated microorganisms. In one embodiment of the invention,microorganisms suitable with the present invention may be isolated andidentified as useful microorganism strains by growing strains in thepresence of a mixture of amide containing amino acids, or derivativesthereof. The microorganism can be isolated or selected or obtained fromknown sources or can be screened from future sources based on theability to detoxify a mixture of nitriles or a mixture of nitrile andamide compounds or a mixture of amides to the corresponding amide and/oracid after multiple induction according to the present invention. Inlight of the disclosure provided herein, it would be a matter of routineexperimentation for one of skill in the art to carry out an assay todetermine whether the microorganism was useful according to the presentinvention. For example, in one assay, the presence of nitrile hydrataseor amidase activity can be determined through detection of free ammonia.See Fawcett, J. K. and Scott, J. E., 1960, “A Rapid and Precise Methodfor the Determination of Urea”, J. Clin. Pathol. 13:156-159, which isincorporated herein by reference.

The present invention beneficially provides methods for cultivatingmicroorganisms, particularly nitrile hydratase producing microorganisms.In certain embodiments, the invention is directed to methods forinducing a desired enzyme activity in microorganisms, such as nitrilehydratase producing enzymes. Preferentially, the method comprisesculturing a nitrile hydratase producing microorganism in a mediumcomprising one or more amide containing amino acids, or derivativesthereof. In one embodiment, the invention provides a method for inducingnitrile-detoxification activity using a medium supplemented with amidecontaining amino acids, or derivatives thereof, which preferably includeasparagine, glutamine or a combination thereof. More particularly, themethod comprises culturing the microorganism in the medium andoptionally collecting the cultured microorganisms or enzymes produce bythe microorganisms.

The microorganisms can be cultured and harvested according to methodsuseful for achieving optimal biomass. In certain embodiments, such aswhen cultured on agar plates, the microorganisms can be cultured for aperiod of at least about 24 but generally less than six days. Whencultured in a fermentor, the microorganisms are preferably cultured in aminimal medium for a period of about 1 hour to about 48 hours, about 1hour to about 20 hours, or about 16 hours to about 23 hours. If a largerbiomass is desired, the microorganisms can be cultured in the fermentorfor longer time periods. At the end of the culture period, the culturedmicroorganisms are typically collected and concentrated, for example, byscraping, centrifuging, filtering, or any other method known to thoseskilled in the art.

In specific embodiments, the microorganisms can be cultured underfurther specified conditions. For example, culturing is preferablycarried out at a pH between about 3.0 and about 11.0, more preferablybetween about 6.0 and about 8.0. The temperature at which culturing isperformed is preferably between about 4° C. and about 55° C., morepreferably between about 15° C. and about 37° C. Further, the dissolvedoxygen tension is preferentially between about 0.1% and 100%, preferablybetween about 4% and about 80%, and more preferably between about 4% andabout 30%. The dissolved oxygen tension may be monitored and maintainedin the desired range by supplying oxygen in the form of ambient air,pure oxygen, peroxide, and/or other compositions which liberate oxygen.

It is also possible according to the present invention to separate thesteps of microorganism growth and enzyme activity induction. Forexample, it is possible according to the invention to grow one or moremicroorganisms on a first medium that does not include supplementationnecessary to induce enzyme activity. Such can be referred to as a growthphase for the microorganisms. In a second phase (i.e., an inductionphase), the cultured microorganisms can be transferred to a secondmedium comprising supplementation necessary to induce enzyme activity.Such second medium would preferentially comprise the amide containingamino acids, or derivatives thereof, as described herein.

Similarly, the induction supplements can be added at any time duringcultivation of the desired microorganisms. For example, the media can besupplemented with amide containing amino acids, or derivatives thereof,prior to beginning cultivation of the microorganisms. Alternately, themicroorganisms could be cultivated on a medium for a predeterminedamount of time to grow the microorganism, and amide containing aminoacids, or derivatives thereof, could be added at one or morepredetermined times to induce the desired activity in themicroorganisms. Moreover, the amide containing amino acids, orderivatives thereof, could be added to the growth medium (or to aseparate mixture including the previously grown microorganisms) toinduce the desired activity in the microorganisms after the growth ofthe microorganisms is complete.

As noted above, the methods of the invention are particularly useful forinducing a desired enzyme activity. Many types of microorganisms,including those described herein, are capable of producing a variety ofenzymes having a variety of activities. As is generally understood inthe art, the type of enzyme activity induced in microorganismcultivation can vary depending upon the strain of microorganism used,the method of growth used, and the supplementation used with the growthmedia. The present invention surprisingly allows for induction of avariety of enzyme activities through the use of amide containing aminoacids, or derivatives thereof. In preferred embodiments, the presentinvention provides for induction of one or more enzymes selected fromthe group consisting of nitrile hydratase, amidase, and asparaginase I.In specific embodiments, such enzymes are induced by culturing one ormore bacteria from the genus Rhodococcus in a medium comprising one ormore amide containing amino acids, or derivatives thereof.

In particular embodiments, the invention allows for the simultaneousinduction of both nitrile hydratase and amidase. This is particularlyuseful for industrial applications, such as the treatment ofnitrile-containing waste streams. Such treatment requires a firsttreatment to convert nitriles to amides and a second treatment toconvert amides to acids. The ability to simultaneously produce nitrilehydratase and amidase would remove the need to separately prepare theenzymes and would essentially allow for a single treatment step.

In further embodiments, the invention particularly provides for theinduction of asparaginase I activity. Surprisingly, it has beendiscovered that asparaginase I activity can be induced in Rhodococcusrhodochrous, DAP 96622 (Gram positive), or Rhodococcus sp., DAP 96253(Gram positive), in medium supplemented with amide containing aminoacids, or derivatives thereof. Other strains of Rhodococcus can alsopreferentially be used according to this embodiment of the invention.Yet other strains capable of producing asparaginase I includePseudomonas chloroaphis (ATCC 43051) (Gram positive), Pseudomonaschloroaphis (ATCC 13985) (Gram positive), Rhodococcus erythropolis (ATCC47072) (Gram positive), and Brevibacterium ketoglutamicum (ATCC 21533)(Gram positive), Thus, the present invention is further beneficial inthat it allows for the induction of asparaginase I activity inGram-positive bacteria.

The desired activity (e.g., nitrile hydratase, amidase, or asparaginaseI activity) of the harvested microorganisms, once induced according tothe methods described herein, can beneficially be stabilized. Commercialuse of enzymes for the treatment of waste water, as well as othercommercial uses of various enzymes, is generally limited by theinstability of the induced activity. For examples, fresh cells willtypically lose at least 50% of their initial activity within 24 hours ata temperature of 25° C. Thus, when cells are to be used as a catalyst,the cells must be made at the time of need and cannot be stored forfuture use. Nitrile hydratase activity can be stabilized throughaddition of nitrile containing compounds; however, this againnecessitates the use of undesirable, hazardous chemicals. The presentinvention again solves this problem. For example, cells having inducednitrile hydratase activity can be stabilized according to the presentinvention, without the need for hazardous chemicals, such that the cellshave a viable enzyme activity for a time period of up to one year. Thus,the present invention stabilizes enzymes, or microorganisms capable ofproducing such enzymes, such that the practical activity of the inducedactivity is extended well beyond the typical period of useful activity.

In one embodiment, such stabilization is provided by addition of one ormore amide containing amino acids, or derivatives thereof. The amidecontaining amino acids, or derivatives thereof, can be added to themicroorganisms at the time of culturing the microorganisms or can beadded to a mixture comprising enzyme activity induced microorganisms,cells, or subunit enzymes. Any amide containing amino acids, orderivatives thereof, as previously described herein can be used forstabilization of induced activity according to this embodiment of theinvention.

In further embodiments, stabilization can be provided, according to theinvention, by immobilizing the cultured microorganism, or cellstherefrom. For example, cells harvested from the microorganisms, enzymesharvested from the microorganisms, or the induced microorganismsthemselves, can be immobilized to a substrate as a means to stabilizethe induced activity. In certain embodiments, the enzymes, cells, ormicroorganisms are at least partially entrapped in the substrate.

Any substrate generally useful for affixation of cells, enzymes, ormicroorganisms can be used according to the invention. In oneembodiment, the substrate comprises alginate, or salts thereof. Alginateis a linear copolymer with homopolymeric blocks of (1-4)-linkedβ-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues,respectively, covalently linked together in different sequences orblocks. The monomers can appear in homopolymeric blocks of consecutiveG-residues (G-blocks), consecutive M-residues (M-blocks), alternating Mand G-residues (MG-blocks), or randomly organized blocks. In a preferredembodiment, calcium alginate is used as the substrate. Particularlypreferred is calcium alginate that has been cross-linked, such as withpolyethyleneimine, to form a hardened calcium alginate substrate.Further description of such immobilization techniques can be found inBucke, C. (1987), “Cell Immobilization in Calcium Alginate”, Methods inEnzymology, volume 135, Part B (ed. K. Mosbach) pp. 175-189, which isincorporated herein by reference. The stabilization effect ofimmobilization using polyethyleneimine cross-linked calcium alginate isillustrated in FIG. 1, which is further described below in Example 2.

In another embodiment, the substrate comprises an amide-containingpolymer. Any polymer comprising one or more amide groups could be usedaccording to the invention. In one preferred embodiment, the substratecomprises a polyacrylamide polymer. The stabilization effect ofimmobilization using polyacrylamide is illustrated in FIG. 2, which isfurther described below in Example 3.

Stabilization can further be achieved, according to the invention,through cross-linking. For example, cells harvested from an inducedmicroorganism can be chemically cross-linked to form agglutinations ofcells. In one preferred embodiment, cells harvested from an inducemicroorganism are cross-linked using glutaraldehyde. For example, cellscan be suspended in a mixture of de-ionized water and glutaraldehydefollowed by addition of polyethyleneimine until maximum flocculation isachieved. The cross-linked cells (typically in the form of particlesformed of a number of cells) can be harvested by simple filtration.Further description of such techniques is provided in Lopez-Gallego, etal., “Enzyme Stabilization by Glutaraldehyde Crosslinking of AbsorbedProteins on Aminated Supports”, J. Biotechnol. 119:70-75, which isincorporated herein by reference. The stabilization effect ofglutaraldehyde cross-linking is illustrated in FIG. 4, which is furtherdescribed below in Example 5.

In another embodiment, the microorganisms can be encapsulated orimmobilized rather than allowed to remain in the classic Brownianmotion. Such immobilization facilitates collection, retention, and reuseof the microorganisms and generally comprises affixation of themicroorganisms to a substrate. Such affixation can also facilitatestabilization of the microorganisms, as described above, or may besolely to facilitate ease of handling of the induced microorganisms orenzymes.

The microorganisms can be immobilized by any method generally recognizedas useful for immobilization of microorganisms, such as sorption,electrostatic bonding, covalent bonding, and the like. Generally, themicroorganisms are immobilized on a solid support which aids in therecovery of the microorganisms from a mixture or solution, such as adetoxification reaction mixture. Suitable solid supports include, butare not limited to granular activated carbon, compost, wood residueproducts, (e.g., wood chips, wood, nuggets, shredded pallets or trees),metal or metal oxide particles (e.g., alumina, ruthenium, iron oxide),ion exchange resins, DEAE cellulose, DEAE-SEPHADEX® polymer, ceramicbeads, cross-linked polyacrylamide beads, cubes, prills, or other gelforms, alginate beads, κ-carrageenan cubes, as well as solid particlesthat can be recovered from the aqueous solutions due to inherentmagnetic ability. The shape of the catalyst is variable (in that thedesired dynamic properties of the particular entity are integrated withvolume/surface area relationships that influence catalyst activity). Inpreferred embodiments, the induced microorganism is immobilized inalginate beads that have been cross-linked with polyethyleneimine or isimmobilized in a polyacrylamide-type polymer.

In specific embodiments, the invention provides methods for detoxifyinga mixture of nitrites by converting the nitrites to the correspondingamides or acids. In one embodiment, the method comprises applying aculture of nitrile degrading microorganisms to a mixture of nitrites andmultiply inducing the microorganisms with a mixture of amide containingamino acids, or derivatives thereof, for a sufficient amount of time toconvert the nitrites to the corresponding amides. Alternatively, themethod comprises applying multiply induced microorganisms to a mixtureof nitrites for a sufficient amount of time to convert the nitrites tothe corresponding amides.

When the microorganisms are applied to a waste stream, themicroorganisms may be growing (actively dividing) or resting (notactively dividing). When the method entails application of an activelygrowing culture of microorganisms, the application conditions arepreferably such that bacterial growth is supported or sustained. Whenthe method entails application of a culture of microorganisms which arenot actively dividing, the application conditions are preferably suchthat enzymatic activities are supported.

In specific embodiments, the present invention can be used to treatwaste streams from a production plant having waste that typicallycontains high concentrations of nitrites, cyanohydrin(s), or otherchemicals subject to enzymatic degradation. For example, the inventionprovides a detoxification method to detoxify a mixture of nitrilecompounds or a mixture of nitrile and amide compounds in an aqueouswaste stream from a nitrile production plant. Further, the presentinvention could be used for treatment of waste streams in the productionof acrylonitrile butadiene styrene (ABS), wherein acrylonitrile is usedin the production of the ABS.

The present invention also provides a biofilter that can be used in thedetoxification of mixtures of nitrile compounds, mixtures of nitrile andamide compounds and mixtures of amide compounds in effluents such asair, vapors, aerosols, and water or aqueous solutions. For example, ifvolatile nitrile compounds are present, the volatiles may be strippedfrom solid or aqueous solution in which they are found and steps shouldbe carried out in such a way that the volatiles are trapped in abiofilter. Once trapped, the volatiles can be detoxified with a pureculture or an extract of a microorganism, as described herein.

The present invention also provides for kits comprising a culture of amicroorganism which has been multiply induced and is able to detoxify amixture of nitrile compounds, a mixture of nitrile and amide compounds,or a mixture of amide compounds. The microorganism can be activelydividing or lyophilized and can be added directly to an aqueous solutioncontaining the nitrile and/or amide compounds. In a preferredembodiment, the kit comprises an induced lyophilized sample. Themicroorganism also can be immobilized onto a solid support, as describedherein. Other kit components can include, for example, a mixture ofinduction supplements, as described herein, for induction of themicroorganisms, as well as other kit components, such as vials,packaging components, and the like, which are known to those skilled inthe art.

EXPERIMENTAL

The present invention will now be described with specific reference tovarious examples. The following examples are not intended to be limitingof the invention and are rather provided as exemplary embodiments.

Example 1 Nitrile Hydratase and Amidase Induction

The induction of nitrile hydratase activity and amidase activity inRhodococcus sp., strain DAP 96253, was evaluated using multiple types ofinducers (1000 ppm). Three different samples were cultured in YEMEAmedium containing 10 ppm cobalt and 7.5 g/L urea and supplemented withacrylonitrile, asparagine, or glutamine. The specific nitrile hydrataseactivity and the specific amidase activity in each sample was evaluated,and the results are provided below in Table 1, with activities providedin units/mg cdw (cell dry weight). One unit of nitrile hydrataseactivity relates to the ability to convert 1 μmol of acrylonitrile toits corresponding amide per minute, per milligram of cells (dry weight)at a of pH 7.0 and a temperature of 30° C. One unit of amidase activityrelates to the ability to convert 1 μmol of acrylamide to itscorresponding acid per minute, per milligram of cells (dry weight) pH of7.0 and a temperature of 30° C. TABLE 1 Nitrile Hydratase ActivityAmidase Activity Supplement (Units/mg cdw) (Units/mg cdw) Acrylonitrile162.23 7.59 Asparagine 170.50 13.24 Glutamine 173.45 10.39

As seen in Table 1, the use of asparagine or glutamine as an inducer fornitrile hydratase activity exceeds the ability of acrylonitrile toinduce such activity. Moreover, the use of glutamine as an inducerresulted in amidase activity approximately 37% greater than the amidaseactivity resulting from the use of acrylonitrile, and asparagineprovided approximately 74% greater activity than acrylornitrile.

Example 2 Stabilization of Nitrile Hydratase Activity Using CalciumAlginate Immobilization

Testing was performed to evaluate the relative stability of cellsinduced for nitrile hydratase activity using asparagine in the culturemedium. Rhodococcus sp., strain DAP 96253, was cultured using a standardculture medium alone or supplemented with asparagine. Cells wererecovered from the culture and immobilized in calcium alginate beads(2-3 mm diameter). To prepared the beads, 25 g of a 4% sodium alginatesolution (1 g sodium alginate in 24 ml of 5 mM TRIS-HCl-pH 7.2) wasprepared, and 25 mg of sodium meta-periodate was dissolved therein(stirred at 25° C. for 1 hr or until all alginate has dissolved). Thecells for immobilization were suspended in 50 mM TRIS-HCl to a finalvolume of 50 ml, and the cell solution was added to the alginate mixturewhile stirring. Beads were formed by extruding the mixture through a 27Ghypodermic needle into 500 ml of 0.1M CaCl₂. The beads were cured for 1hr in the CaCl₂ solution and washed with water.

Four samples were prepared for evaluation: Sample 1—beads formed withcells cultured without asparagine but with asparagine added to themixture including the beads; Sample 2—beads formed with cells culturedwith asparagine and having asparagine added to the mixture including thebeads; Sample 3 —beads formed with cells cultured with asparagine andhaving a mixture of acrylonitrile and acetonitrile added to the mixtureincluding the beads; and Sample 4—beads formed with cells cultured withacrylonitrile and acetonitrile and having asparagine added to themixture including the beads. In samples 3 and 4, acrylonitrile andacetonitrile were added in a concentration of 500 ppm each. In each ofsamples 1-4, asparagine was added at 1000 ppm.

The immobilized cells were maintained for a time of about 150 hours andperiodically evaluated for the remaining nitrile hydratase activity. Theresults of the test are illustrated in FIG. 1. For evaluation ofstabilized activity, equivalent amounts of cells were tested, and theactivity of an equivalent aliquot of whole cells at time 0 was set as100%. Equivalent aliquots of catalyst were incubated at the appropriatetemperature. At the appropriate times, an entire aliquot was removedfrom incubation and the enzyme activity determined. For the first 10hours samples were evaluated every 2 hours. From 10-60 hours sampleswere evaluated every 4 hours and thereafter, samples were evaluatedevery 12 hours.

As seen in FIG. 1, immobilization of induced cells in calcium alginateprovides stabilization of nitrile hydratase activity that is verysimilar to the level of stabilization achievable using hazardous nitrilecontaining compounds but without the disadvantages (e.g., health andregulatory issues).

Example 3 Stabilization of Nitrile Hydratase Activity UsingPolyacrylamide Immobilization

Testing was performed to evaluate the relative stability of cellsinduced for nitrile hydratase activity using asparagine in the culturemedium. Rhodococcus sp., strain DAP 96253, was cultured using a standardculture medium supplemented with asparagine. Cells were recovered fromthe culture and immobilized in cross-linked polyacrylamide cubes (2.5mm×2.5 mm×1 mm). The polyacrylamide solution was prepared, and thedesired loading of cells was added. The polyacrylamide with the cellswas cross-linked to form a gel, which was cut into the noted cubes. Nofurther known stabilizers were added to the polyacrylamide. Two sampleswere prepared for evaluation: Sample 1—cubes with low cell load(prepared with suspension comprising 1 g of cells per 40 mL of cellsuspension); and Sample 2—cubes with high cell load (prepared withsuspension comprising 4 g of cells per 40 mL of cell suspension).

The immobilized cells were maintained for a time of about 150 days andperiodically evaluated for the remaining nitrile hydratase activity. Theresults of the test are illustrated in FIG. 2. For evaluation ofstabilized activity, equivalent amounts of cells were tested, and theactivity of an equivalent aliquot of whole cells at time 0 was set as100%. Equivalent aliquots of catalyst were incubated at the appropriatetemperature. At the appropriate times, an entire aliquot was removedfrom incubation and the enzyme activity determined. For the first 10hours samples were evaluated every 2 hours. From 10-60 hours sampleswere evaluated every 4 hours. From 5 days to 40 days samples wereevaluated every 12 hours. From 40 to 576 days, samples were evaluated onaverage every 10 days.

As seen in FIG. 2, cells stabilized using polyacrylamide maintainedactivity as much as 150 hours after induction. Moreover,polyacrylamide-immobilized cells loaded at a low concentration stillexhibited 50% of the initial activity at about 45 hours after induction,and polyacrylamide-immobilized cells loaded at a high concentrationstill exhibited 50% of the initial activity at about 80 hours afterinduction.

Example 4 Stabilization of Nitrile Hydratase Activity Using CalciumAlginate or Polyacrylamide Immobilization

Testing was performed to evaluate the relative stability of cellsinduced for nitrile hydratase activity using asparagine in the culturemedium. The testing specifically compared the stabilization provided byimmobilization in polyacrylamide or calcium alginate. Rhodococcus sp.,strain DAP 96622, was cultured using a standard culture mediumsupplemented with asparagine to induce nitrile hydratase activity. Cellswere recovered from the culture for immobilization.

Test Sample 1 was prepared by immobilizing the asparagine induced cellsin polyacrylamide cubes (2.5 mm×2.5 mm×. 1 mm) using the methoddescribed in Example 3. As a comparative, cells separately induced usingacrylonitrile were also immobilized in polyacrylamide cubes forevaluation.

Test Sample 2 was prepared by immobilizing the asparagine induced cellsin calcium alginate beads (2-3 mm diameter) using the method describedin Example 2. As a comparative, one sample was prepared using actualnitrile containing waste water as the inducing supplement (denotedNSB/WWCB). A second comparative was prepared using, as the inducer, asynthetic mixture containing the dominant nitriles and amides present inan acrylonitrile production waste stream (also including ammoniumsulfate and expressly excluding hydrogen cyanide) (denoted FC w/AMS w/oHCN).

The immobilized cells were maintained for a time of about 576 days andperiodically evaluated for the remaining nitrile hydratase activity. Theresults of the test are illustrated in FIG. 3. For evaluation ofstabilized activity, equivalent amounts of cells were tested. Theactivity of an equivalent aliquot of whole cells at time 0 was set as100%. Equivalent aliquots of catalyst were incubated at the appropriatetemperature. At the appropriate times, an entire aliquot was removedfrom incubation and the enzyme activity determined. For the first 10hours samples were evaluated every 2 hours. From 10-60 hours sampleswere evaluated every 4 hours. From 5 days to 40 days samples wereevaluated every 12 hours. From 40 to 576 days, samples were evaluated onaverage every 10 days.

Example 5 Stabilization of Nitrile Hydratase Activity UsingGlutaraldehyde Immobilization

Testing was performed to evaluate the relative stability of cellsinduced for nitrile hydratase activity using asparagine in the culturemedium. The testing specifically compared the stabilization provided byimmobilization via glutaraldehyde cross-linking. Rhodococcus sp., strainDAP 96253, and Rhodococcus rhodochrous, strain DAP 96622, wereseparately cultured using a standard culture medium supplemented withasparagine to induce nitrile hydratase activity. Cells were recoveredfrom the culture and cross-linked using glutaraldehyde, as describedherein.

The immobilized cells were maintained for a time of about 576 days andperiodically evaluated for the remaining nitrile hydratase activity. Theresults of the test are illustrated in FIG. 4. For evaluation ofstabilized activity, equivalent amounts of cells were tested. Theactivity of an equivalent aliquot of whole cells at time 0 was set as100%. Equivalent aliquots of catalyst were incubated at the appropriatetemperature. At the appropriate times, an entire aliquot was removedfrom incubation and the enzyme activity determined. For the first 10hours samples were evaluated every 2 hours. From 10-60 hours sampleswere evaluated every 4 hours. From 5 days to 40 days samples wereevaluated every 12 hours. From 40 to 576 days, samples were evaluated onaverage every 10 days.

As seen in FIG. 4, both strains immobilized via glutaraldehydecross-linking exhibited somewhat less initial activity in comparison toother stabilizations methods described above. However, both strainsimmobilized via glutaraldehyde cross-linking exhibited excellentlong-term stabilization maintaining as much as 65% activity after 576days.

Example 6 Effect of Asparagine and Glutamine on Growth of NitrileHydratase Producing Microorganisms

The relative growth of various nitrile hydratase producingmicroorganisms was evaluated. All strains were grown on YEMEA mediumcontaining 7.5 g/L of urea and 10 ppm cobalt (provided as cobaltchloride) supplemented with asparagine (ASN), glutamine (GLN), or bothasparagine and glutamine. The asparagine and glutamine were added 3.8mM. Growth temperature was in the range of 26° C. to 30° C. Growth wasevaluated by visual inspection and graded on the following scale: (−)meaning no detectable growth; (+/−) meaning scant growth; (+) meaninglittle growth; (++) meaning good growth; (+++) meaning very good growth;and (++++) meaning excellent growth. The results are provided below inTable 2. TABLE 2 Growth Medium Growth Supplementation Strain ATCC #Temp. (° C.) ASN GLN ASN + GLN Pseudomonas 43051 30 + − + chloroaphisPseudomonas 13985 26 + + ++ chloroaphis Brevibacterium 21533 30 + + +ketoglutaricum Rhodococcus 47072 26 ++ ++ +++ erythropolis Rhodococcussp. 55899 30 ++++ ++++ ++++ DAP 96253 Rhodococcus 55898 26 ++++ ++++++++ rhodochrous DAP 96622

Example 7 Effect of Asparagine and Glutamine on Nitrile Hydratase andAmidase Production

The induction of nitrile hydratase production and amidase production invarious nitrile hydratase producing microorganisms was evaluated. Allstrains were grown on YEMEA medium containing 7.5 g/L of urea and 10 ppmcobalt (provided as cobalt chloride) supplemented with asparagine (ASN),glutamine (GLN), or both asparagine and glutamine. The asparagine andglutamine were added 3.8 mM. As a comparative, enzyme production with nosupplementation was also tested. Growth temperature was in the range of26° C. to 30° C. The nitrile hydratase level in Units per mg of cell dryweight was evaluated, and the results are provided in Table 3. Theamidase level in Units per mg of cell dry weight was evaluated, and theresults are provided in Table 4. TABLE 3 Nitrile Hydratase Level(Units/mg cdw) Based on Growth Medium Growth Supplementation Temp. ASN +Strain ATCC # (° C.) ASN GLN GLN None Pseudomonas 43051 30 28 No 45 49chloroaphis growth Pseudomonas 13985 26 14 0 8 30 chloroaphisBrevibacterium 21533 30 30 37 42 34 ketoglutaricum Rhodococcus 47072 2648 42 55 55 erythropolis Rhodococcus sp. 55899 30 155 135 152 82 DAP96253 Rhodococcus 55898 26 158 160 170 63 rhodochrous DAP 96622

TABLE 4 Amidase Level Growth (Units/mg cdw) Based on ATCC Temp. GrowthMedium Supplementation Strain # (° C.) ASN GLN ASN + GLN NonePseudomonas 43051 30 0 No 0 0 chloroaphis growth Pseudomonas 13985 26 140 8 4 chloroaphis Brevibacterium 21533 30 0 0 3 2 ketoglutaricumRhodococcus 47072 26 9 14 6 2 erythropolis Rhodococcus 55899 30 13 7 104 sp. DAP 96253 Rhodococcus 55898 26 10 6 12 5 rhodochrous DAP 96622

Example 8 Effect of Asparagine and Glutamine on Asparaginase IProduction

The induction of asparaginase I production in various nitrile hydrataseproducing microorganisms was evaluated. All strains were grown on YEMEAmedium containing 7.5 g/L of urea and 10 ppm cobalt (provided as cobaltchloride) supplemented with asparagine (ASN), glutamine (GLN), or bothasparagine and glutamine. The asparagine and glutamine were added 3.8mM. As a comparative, enzyme production was also evaluated withsupplementation was with acrylonitrile (AN), acrylamide (AMD) oracrylonitrile and acrylamide. Growth temperature was in the range of 26°C. to 30° C. The asparaginase I level in Units per mg of cell dry weightwas evaluated, and the results are provided in Table 5. TABLE 5 GrowthAsparaginase I Level (Units/mg cdw) Based Temp. on Growth MediumSupplementation Strain ATCC # (° C.) AN AMD AN/AMD ASN GLN ASN/GLNPseudomonas 43051 30 — — — 18.4 No 18.7 chloroaphis Growth Pseudomonas13985 26 2 0 3 0 O 1 chloroaphis Brevibacterium 21533 30 14.6 15.4 13.619.1 20.3 17.8 ketoglutaricum Rhodococcus 47072 26 — 0 0 1 2 0erythropolis Rhodococcus 55899 30 7.8 2 7.4 12.5 11.1 13.9 sp. DAP 96253Rhodococcus 55898 26 8.2 7.8 10.1 12.3 10 13.8 rhodochrous DAP 96622

Example 9 Induction of Asparaginase I Activity in Rhodococcus sp. DAP96253 Cells

Rhodococcus sp. DAP 96253 were grown using biphasic medium as the sourceof inoculum for a 20 liter fermentation. The supplemental addition ofmedium/carbohydrate (either YEMEA, dextrose or maltose) was made to themodified R2A medium, containing cottonseed hydrolysate substituted forthe Proteos Peptone 3 (PP3). Asparagine (0.15M solution) was added at acontinuous rate of 1000 μl/min beginning at t=10 h. At the end of thefermentation run, 159 units per milligram cell dry weight ofacrylonitrile specific nitrile hydratase, 22 units of amidase permilligram cell dry weight, and 16 g/l cell packed wet weight wereproduced. The amount of various enzymes produced is provided in FIG. 5.As can be seen therein, 159 units of nitrile hydratase, 22 units ofacrylamidase, and 16 units of asparaginase I per milligram cell dryweight was produced by the DAP 96253 cells.

Example 10 Effect of Media Composition on Asparaginase I Production inRhodococcus sp. DAP 96253 Cells

Testing was performed to evaluate the effect on asparaginase I activitybased upon the inducer used. In particular, testing was performed usingasparagine, glutamine, succinonitrile, and isovaleronitrile as inducers(all added at 1000 ppm each). As can be seen in Table 6, asparagine wasable to induce asparaginase I activity of 24.6 units/mg cell dry weight.Glutamine or succinonitrile also showed an ability to induceasparaginase I activity. Higher asparaginase I activity was obtainedwhen maltose was added to YEMEA. The inclusion of Cobalt (5-50 ppm) inthe medium also resulted in improvements when combined with eitherglucose or maltose. TABLE 6 Asparaginase I levels in Rhodococcus sp. DAP96253 Grown in Medium with Carbohydrate Supplement YEMEA - YEMEA -Glucose Maltose Without With Without With Inducer Cobalt Cobalt CobaltCobalt Asparagine 5.3 6.5 8.7 24.6 Glutamine 1.5 1.9 9.3 8.1Succinonitrile 6.5 8.5 11.0 10.0 Isovaleronitrile 3.5 2.9 6.8 7.0

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions. Therefore, it is to be understood that theinventions are not to be limited to the specific embodiments disclosedand that modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A method for inducing desired enzyme activity in a nitrile hydrataseproducing microorganism comprising culturing the nitrile hydrataseproducing microorganism in a medium comprising at least about 50 ppm ofone or more amide containing amino acids or derivatives thereof.
 2. Themethod of claim 1, wherein the desired enzyme activity is selected fromthe group consisting of nitrile hydratase activity, amidase activity,asparaginase I activity, and combinations thereof.
 3. The method ofclaim 1, wherein the nitrile hydratase producing microorganism comprisesbacteria selected from the group consisting of genus Rhodococcus, genusBrevibacterium, genus Pseudomonas, genus Pseudonocardia, genus Nocardia,and combinations thereof.
 4. The method of claim 1, wherein the nitrilehydratase producing microorganism comprises bacteria selected from thegroup consisting of Rhodococcus rhodochrous DAP 96622, Rhodococcus sp.DAP 96253, and combinations thereof.
 5. The method of claim 1, whereinthe one or more amide containing amino acids are selected from the groupconsisting of asparagine, glutamine, or combinations thereof.
 6. Themethod of claim 1, wherein the one or more amide containing amino acidsare present in a concentration of about 50 ppm to about 5000 ppm.
 7. Themethod of claim 1, wherein the medium is free of any nitrile containingcompounds.
 8. The method of claim 1, further comprising immobilizing themicroorganism.
 9. The method of claim 1, wherein the induced nitrilehydratase producing microorganism has an enzyme activity greater than orequal to the activity of the same enzyme when induced using a nitrilecontaining compound.
 10. The method of claim 1, wherein the inducednitrile hydratase producing microorganism has an enzyme activity that isat least 5% greater than the activity of the same enzyme when inducedusing a nitrile containing compound.
 11. The method of claim 1, whereinthe medium further comprises cobalt.
 12. The method of claim 1, whereinthe medium further comprises urea.
 13. A method for stabilizing desiredactivity in an enzyme or a microorganism capable of producing the enzymecomprising contacting the enzyme or microorganism capable of producingthe enzyme with a composition comprising at least about 50 ppm of one ormore amide containing amino acids, or derivatives thereof.
 14. Themethod of claim 13, wherein the one or more amide containing amino acidsare selected from the group consisting of asparagine, glutamine, andcombinations thereof.
 15. The method of claim 13, wherein the one ormore amide containing amino acids are present in a concentration ofabout 50 ppm to about 5000 ppm.
 16. The method of claim 13, wherein thedesired activity of the enzyme or the microorganism capable of producingthe enzyme is stabilized such that the desired activity after a time ofat least 30 days at a temperature of 25° C. is maintained at a level ofat least about 50% of the initial activity exhibited by the enzyme orthe microorganism capable of producing the enzyme.
 17. A method forstabilizing nitrile hydratase comprising immobilizing the nitrilehydratase or microorganism capable of producing the nitrile hydratasewith a substrate such that the activity after a time of at least 30 daysat a temperature of 25° C. is maintained at a level of at least about50% of the initial activity exhibited by the nitrile hydratase or themicroorganism capable of producing the nitrile hydratase.
 18. The methodof claim 17, wherein the substrate is selected from the group consistingof alginate and amide-containing polymers.
 19. The method of claim 18,wherein the amide-containing polymer comprises polyacrylamide.
 20. Themethod of claim 17, wherein the immobilizing comprises cross-linkingcells from the microorganism with glutaraldehyde.
 21. The method ofclaim 17, wherein the microorganism comprises bacteria selected from thegenus Rhodococcus, genus Brevibacterium, genus Pseudomonas, genusPseudonocardia, genus Nocardia, and combinations thereof.